Gene Therapy Mutations in RPGR are responsible for 15-20% of all inherited retinal degeneration. We previously worked with other groups for gene replacement therapy in RPGR-dog models. In collaboration with Dr. Zhijian Wu, we initiated and completed long-term pre-clinical studies for RPGR disease using mouse models and AAV-based gene therapy. Another project is on CEP290-LCA. The full-length CEP290 cDNA is large and not deliverable by AAV because of the packaging limit. In addition, CEP290-LCA is caused by partial loss of function, and overexpression of CEP290 leads to cellular toxicity. We are therefore evaluating whether different domains of the CEP290 protein (delivered by AAV) can rescue retinal phenotype in rd16 or rd16/Nrl-/- mice that we previously discovered as models of CEP290-LCA. LCA Disease Modeling and Rescue Strategies using Stem Cells Reprogramming of somatic cells to pluripotency allows de novo differentiation of PRs and model retinal disease in a dish using patients own cells and test various paradigms for therapy. As stated earlier, our focus is on LCA for designing interventions. Mutations in CEP290 cause a wide spectrum of clinical phenotypes, including LCA and Joubert syndrome related disorders (JSRD). To elucidate mechanisms of vision-centric versus syndromic CEP290 disease, we produced fibroblast cell lines and later iPSC lines from three LCA and two JSRD patients and their respective familial controls. Interestingly, iPSC-derived optic vesicles from CEP290-LCA patients revealed less developed PR cilia compared to controls. Our studies thus recapitulate the pathogenic changes in CEP290-LCA-specific human iPSC-derived 3D retina and should serve as a useful model to test treatment strategies. CRX mutations can also have different clinical presentations; We previously reported that two CRX mutations, p.K74N and p.I138fs48, have differential impact of NRL function and are associated with dominant LCA. To interventions for dominant CRX-LCA, we have generated iPSC lines from the two patients and one of their unaffected parents. We are generating optic vesicles and photoreceptors from these lines and examining if the mutations cause any aberrant phenotypes. Drug Discovery Small molecules that can rescue IRD-associated changes in PRs have the potential to target retinal degeneration caused by multiple distinct genetic defects. We are using two approaches to identify such molecules, as follows: Small molecule screens using retinal organoids from iPSC lines derived from mouse models of and patients with CEP290- and CRX-LCA As elaborated in (Kaewkhaw et al. 2016), we have initiated small molecule screenings to rescue phenotypes observed in mutant retinal organoids. Differentiation of neural retina in organoid cultures from human iPSC lines takes over 200 days. Hence, screening studies are initially being performed using organoid cultures from a mouse model of CEP290-LCA. Pathway-based Drug Discovery: Dysfunction or death of photoreceptors is the common final outcome of most genetic defects observed in retinopathies. We hypothesized that cellular pathways adversely impacted by disease-associated variants trigger only a small number of common pre-apoptotic signals (PAS) and that PAS would be good targets for drug discovery and development of generalized therapies for most inherited retinal diseases. To identify PAS, we focused on six mouse retinal degeneration mutants: Pde6brd1, Cep290rd16, Aipl1-/-, Pde6brd10, Rpgrip1-/-, and Rds (Prph2rd2), with defects affecting distinct cellular functions and having different rates of photoreceptor cell death. We are currently analyzing the large amount of temporal transcriptome data to identify genes or pathways showing similar dynamics in different mutant photoreceptors. In addition, we plan to analyze distinct subgroups (e.g., Rd1 and Rd10, both having defects in the same gene; or ciliopathy mutants).